Effective mechanisms to maintain cellular homeostasis and combat stress are vital to all living organisms. We study two issues fundamental to all living cells: how to construct an effective response to thermal stress (the ?32-directed heat shock response;HSR), and how to maintain homeostasis in discrete compartments of the cell (the ?E-directed envelope stress response). Our future work will address 3 critical questions: 1) How is homeostatic feedback control of ?32 accomplished? The activity of the HSR is coupled to cellular folding state by a homeostatic feedback control system. Surprisingly, our new studies implicate FtsY, the receptor (SR) for the Signal Recognition Particle (SRP), which is required for insertion of proteins into the inner membrane in this pathway. We will examine the role of SR/SRP proteins in feedback regulation, and test the provocative hypothesis that membrane localization of ?32 plays a regulatory role in this process;possibly by amplifying the length of time ?32 is unavailable for binding RNA polymerase. If these studies implicate the SR/SRP machine in cellular control, they will also provide the basis for a paradigm changing view of these universally conserved GTPases. 2) How are envelope stress signals integrated to activate the ?E response? Envelope stress controls the rate of degradation of RseA, the membrane spanning antisigma factor that negatively regulates ?E, thereby coupling ?E activity to status of the cell envelope. Previously, we thought that this pathway was activated simply by unassembled porins but our recent work suggests that two additional signals are required for activation. We will identify these signals, determine the relationship between the three signals, and reconstitute this regulatory pathway in vitro. These studies will provide critical understanding of how the two cellular compartments communicate. 3) What is the impact of sRNAs in the ?E (and ?32) mediated stress responses? Our recent work and that of others indicates that a major aspect of the ?E response is production of sRNAs that downregulate outer membrane porins. We will identify the remaining ?E sRNAs and test the hypothesis that they function to provide overall surveillance of outer membrane porin content and critical control of the properties of the stress response. Additionally, our preliminary evidence indicates that an sRNA controls DnaK, a regulator of ?32. Therefore, we will also identify ?32-controlled sRNAs and screen for those with regulatory roles. PUBLIC HEALTH RELEVANCE: Project relevance Microbes account for fully half of the world's biomass and are of immense importance to life on earth, for reasons as diverse as performing mineral recycling in our ecosystem, making products of important for biotechnology and detoxification of the environment, and causing disease. In the last decade, the genomic sequences of 444 bacteria have been published and hundreds more are in progress. It is impossible to study all of these bacteria, but we can perform an intensive study of selected bacteria, as the paradigms we develop are widely applicable to all bacteria. We study the regulation and function of two critical responses to stress, in the model organism E. coli, where cutting edge studies are possible. These responses are highly conserved, and both have been implicated in pathogenesis in related organisms. Moreover, one of the strategies we are defining is proving to be a paradigm for responses that orchestrate production of antibiotics, virulence and agents important in environmental cleanup. Finally, our studies may uncover new targets for antibiotics.